Method of designing primers for use in method of detecting target nucleic acid and assay kit

ABSTRACT

A method of designing primers for use in a method of detecting an amplification product by hybridizing it with a probe, the amplification product is amplified from a target nucleic acid with the primers, including placing F3, F2 and F1 regions in this order from a 5′ terminal side and Bc, B2 c  and B1 c  regions in this order from a 3′ terminal side, and additionally an FP region in the region from the F2 to F1 regions and/or a BPc region in the region from the B2 c  to B1 c  regions in the target nucleic acid, determining the respective regions in such a manner that the FP and F2 regions and/or the BPc and B2 c  regions have an unoverlapping region of at least 10 bases or more and overlapping regions of 10 bases or less, and designing the primers according to the regions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based upon and claims the benefit of priority fromprior Japanese Patent Application No. 2006-012889, filed Jan. 20, 2006,the entire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of designing primers foramplification of a target nucleic acid and a method of detecting thetarget nucleic acid by using the primer, and an assay kit for themethod.

2. Description of the Related Art

For detection a nucleic acid chain having a particular nucleotidesequence, the nucleic acid is amplified by using a nucleotide primercomplementary thereto. The amplification product is generally identifiedby electrophoresis or hybridization with a probe complementary to thesequence to be detected in the amplification product. The hybridizationwith a probe has the advantage that it is possible to reconfirm thespecificity of amplification product.

A polymerase chain reaction (PCR) method is generally known as the geneamplification method (see, e.g., Science, 230, pp. 1350-54, 1985).However, the method has problems such as demand for complicatedtemperature control unit and low specificity to templates with mutationonly of several bases.

Recently, a loop-mediated isothermal amplification (LAMP) method hasbeen developed as a gene amplification method demanding no suchcomplicated temperature control (see JP No. 3313358). The LAMP method isa method of amplifying a particular gene region under an isothermalcondition at 60 to 65° C. The LAMP method use primers including an innerprimer pair, an outer primer pair, and optionally loop primer pairs (seeWO No. 02/024902), a strand-displacing polymerase, and a substratenucleotide. The LAMP method gives a greater amount of final product thanthe PCR method. It also has advantages such as simple operation, highspeed, and low cost. Accordingly, the LAMP method is expected to be usedin wider fields.

The PCR products are present in the double-stranded chain structure. Forthis reason, when the PCR product is detected with a probe nucleic acid,its complementary chain of the PCR product inhibit as a competitor tothe probe, so hybridization efficiency has decreased. To solve theproblem, developed was a method of preventing self hybridization of thenucleic acid to be detected by binding a nucleic acid chain to a targetnucleic acid chain in the region excluding the sequence regioncomplementary to the probe thereof (see, e.g., JP-A 6-70799 [KOKAI]).However, even the method does not give sufficient sensitivity.Alternatively developed was a method of decomposing or separating thecomplementary chain in the PCR product. However, the method also hadproblems such as high cost due to use of enzyme, magnetic beads and thelike and complicated operation.

On the other hand, the LAMP product has a single-stranded loop regiontherein. Therefore, it is possible to design the region to bind with aprobe. In this way, it is possible to hybridize a probe with the productefficiently without a step of converting the product into singlestrands. Disclosed were various methods of detecting a LAMPamplification product by using a single-stranded loop region. Forexample, JP-A 2002-272475 (KOKAI) discloses a method of labeling a probehybridizing with the single-stranded loop region with fluorescent dyeand measuring its fluorescent polarization. Alternatively, JP-A2002-345499 (KOKAI) discloses a method of immobilizing the 5′ terminalof a primer hybridizing with a single-stranded loop region on animmobilization carrier and monitoring its coagulation reaction. Yetalternatively, JP-A 2005-143492 (KOKAI) discloses a method ofimmobilizing a probe hybridizing with a single-stranded loop region on asolid phase and detecting the hybridization between the probe and theLAMP amplification product based on the fluorescent or electrochemicalprinciple.

However, for the detection methods using a single-stranded region, thereis no principle of design regions of primers and detection regionsclosed each other. Thus, there exists a need for an improved detectionmethod for detecting the LAMP-amplified product efficiently.

BRIEF SUMMARY OF THE INVENTION

An object of the present invention is to provide a method of designingLAMP primers for accurate detection of LAMP amplification product.

According to one aspect of the present invention, there is provided amethod of designing primers for use in a method of detecting anamplification product by hybridizing it with a probe, the amplificationproduct is amplified from a target nucleic acid with the primers,

on the presupposition that:

the target nucleic acid comprise F3, F2 and F1 regions in this orderfrom a 5′ terminal side, B3c, B2c and B1c regions in this order from a3′ terminal side, as well as an FP region in the region from the F2 toF1 regions and/or a BPc region in the region from the B2c to B1cregions;

the probe represents a probe nucleic acid comprising a nucleotidesequence complementary to a sequence selected from the group consistingof the sequence in the FP and BPc region and the sequences complementarythereto; and

the primers contain an FIP primer comprising the sequence identical tothat of the F2 region at the 3′ terminal side and a sequencecomplementary to that of the F1 region at the 5′ terminal side; an F3primer comprising the sequence identical to that of the F3 region; a BIPprimer comprising a sequence complementary to that of the B2c region atthe 3′ terminal side and the sequence identical to that of the B1cregion at the 5′ terminal side; and a B3 primer comprising a sequencecomplementary to that of the B3c region:

the method comprising setting the respective regions in such a mannerthat the FP and F2 regions and/or the BPc and B2c regions have anunoverlapping region of at least 10 bases or more and overlappingregions of 10 bases or less, and designing the primers according to theregions.

According to another aspect of the present invention, there is provideda method of designing primers for use in a method of detecting asingle-nucleotide mutation by amplifying a target nucleic acid havingthe single-nucleotide mutation with primers and hybridizing theamplification product with a probe,

on the presupposition that:

the target nucleic acid comprise F3, F2 and F1 regions in this orderfrom a 5′ terminal side, B3c, B2c and B1c regions in this order from a3′ terminal side, as well as an FP region having the single-nucleotidemutation in the region from the F2 to F1 regions and/or a BPc regionhaving the single-nucleotide mutation in the region from the B2c to B1cregions;

the probe represents a probe nucleic acid comprising a nucleotidesequence complementary to a sequence selected from the group consistingof the sequence in the FP and BPc region, and the sequencescomplementary thereto; and,

the primers contain an FIP primer comprising the sequence identical tothat of the F2 region at the 3′ terminal side and a sequencecomplementary to that of the F1 region at the 5′ terminal side; an F3primer comprising the sequence identical to that of the F3 region; a BIPprimer comprising a sequence complementary to that of the B2c region atthe 3′ terminal side and the sequence identical to that of the B1cregion at the 5′ terminal side; and a B3 primer comprising a sequencecomplementary to that of the B3c region:

the method comprising setting the F2 region not to overlap thesingle-nucleotide mutation in the FP region and/or the B2c region not tooverlap the single-nucleotide mutation in the BPc region, and designingthe primers according to the regions.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

FIG. 1 is a chart showing the locations of respective regions on atarget nucleic acid;

FIG. 2 is a chart showing the locations of detection regions on a targetnucleic acid;

FIG. 3 is a chart showing the location of a loop primer region on atarget nucleic acid;

FIG. 4 is a chart showing LAMP-amplification intermediate products andprimer-annealing sites;

FIG. 5 is a schematic chart showing four kinds of single-stranded loopregions formed in a LAMP product;

FIG. 6 is a schematic chart showing an F2 region and an FP regionoverlapping each other;

FIG. 7 is a schematic chart showing an LF region and an FP regionoverlapping each other;

FIG. 8 is a table showing the relationship between respective sequenceswhen the inner and loop primers are factors inhibiting the probe nucleicacid;

FIG. 9 is a chart showing the design patterns of respective regionsplaced on a target nucleic acid;

FIG. 10 is a chart showing the design patterns of respective regions inan aspect where single-nucleotide mutation is to be detected;

FIG. 11 is a chart showing an embodiment of detection sequences designedon MTHFR gene;

FIG. 12 is a chart showing the detection result of the pseudopositivesignals caused by inner primers;

FIG. 13 is a chart showing examples of detection sequences designed onMTHFR gene;

FIG. 14 is a chart showing the detection result of the pseudopositivesignals caused by loop primers;

FIG. 15 is a chart showing the relationship between the kind of primeradded in Example 2 and the amplification efficiency and detectionresult;

FIG. 16 is a chart showing hybridization inhibition by a loop primer;

FIG. 17 is a chart showing the relationship between the kind of primeradded in Example 3 and the amplification efficiency and detectionresult;

FIG. 18 is a chart showing an embodiment of respective regions placed ona NAT2 gene;

FIG. 19 is a chart showing amplification results when loop primer isplaced on the single-nucleotide mutation site; and

FIG. 20 is a chart showing the relationship between the kind of primeradded in Example 4 and the amplification efficiency and detectionresult.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that the primer used for amplification of LAMPproduct is a cause of inhibition of and deterioration in reactionefficiency of the hybridization reaction, and completed the presentinvention. It is possible to easily obtain a LAMP amplification productwith higher hybridization efficiency, by using a primer designedaccording to the present invention. It is thus possible to detect aparticular target nucleic acid easily at high accuracy.

Hereinafter, aspects of the present invention will be described indetail. In the present specification, the nucleic acid to be detected iscalled a target nucleic acid, and the nucleotide sequence thereof iscalled target sequence. The solution possibly containing the targetnucleic acid used in the detection method according to the presentinvention will be called a sample solution.

In the present invention, in which the products amplified by LAMP methodare detected after hybridization with a probe nucleic acid, the sequencein the amplification product used in hybridization with the probenucleic acid will be referred to as detection sequence. Accordingly, theprobe nucleic acid has a sequence complementary to the detectionsequence.

The LAMP method is a method of amplifying a target nucleic acid presentin a sample by using primers and a chain-substituting DNA polymerase.There are various methods of detecting the amplification product, butthe present invention relates to a detection method by using a probenucleic acid as described above. Hereinafter, the LAMP method will bedescribed briefly.

As shown in FIG. 1, in the LAMP method, F3, F2 and F1 regions are placedin this order from the 5′ terminal side of a target nucleic acid, andB3c, B2c and B1c regions are placed in this order from the 3′ terminalside thereof. In the method according to the present invention ofdetecting an amplification product by hybridizing it with a probenucleic acid, an FP region is placed additionally in the region from theF2 to F1 regions and a BPc region in the region from the B2c to B1cregions, as shown in FIG. 2. The FP and BPc regions and the regions oftheir complementary chains are the detection regions with which theprobe nucleic acid is hybridized and of which the nucleotide sequencesare detected. The sequence for use in detection by hybridization with aprobe nucleic acid may be just one of the sequences in the FP and BPcregions and sequences complementary thereto, or two or more of them maybe used at the same time. Thus, the sequence of the probe nucleic acidis prepared to have a sequence complementary to the detection sequenceamong the sequences above.

The regions having complementary sequence to those of the F3, F2, and F1regions will be called F3c, F2c, and F1c regions, respectively. Theregions having complementary sequence to those of the B3c, B2c, and B1cregions will be called B3, B2, and B1 regions, respectively. Similarly,the regions having complementary sequence to those of the FP and BPcregions will be called FPc and BP regions. In the present specification,the chains having the F3 region or the like will be called plus chains,while the chains having the F3c region or the like will be called minuschains, for convenience.

A primer used for amplification of nucleic acids in the LAMP methodincludes four kinds of basic primers: (1) an FIP primer having thesequence identical to that of the F2 region in the 3′ terminal side anda sequence complementary to that of the F1 region at the 5′ terminalside; (2) an F3 primer having the sequence identical to that of the F3region; (3) a BIP primer having a sequence complementary to that of theB2c region at the 3′ terminal side and the sequence identical to that ofthe B1c region at the 5′ terminal side; and (4) a B3 primer having asequence complementary to that of the B3c region. Generally, the FIP andBIP primers are called inner primers, while the F3 and B3 primers arecalled outer primers.

The amplification period in the LAMP method can be shortened byoptionally using a primer called a loop primer. In such a case, as shownin FIG. 3, an LF region is placed in the region from the F2 to F1regions and an LBc region is placed in the region from the B2c to B1cregions of a target nucleic acid. These regions will be referred to asloop primer regions. In addition to the four kinds of primers above, aloop primer LFc having a sequence complementary to that of the LF regionand a loop primer LBc having the sequence identical to that of the LBcregion are used. See WO 2002/024902 for details. These loop primers LFcand LBc may be used alone or in combination.

FIG. 4 is a schematic chart showing intermediate amplification productsby the LAMP method. In the LAMP method, dumbbell-shaped intermediateproducts are first produced from the target nucleic acid, as shown inthe figure, and become the initial points in the amplification cycle.The methods of generating the initial point structure and subsequentamplifying are known in the art, and see JP No. 3313358 for details. Asshown in FIG. 4, the intermediate products, which have complementarysequences at both terminals, form loops by self-annealing.

As shown in FIG. 4, the FIP and BIP primers become synthetic initialpoints by annealing respectively on the intermediate products, and thus,the synthetic reaction proceeds from the 3′ terminal side. Theintermediate products having the loop structure function as templatesfor the FIP and BIP primers continuously, allowing progress of thenucleic acid synthesis. As shown in the figure, the loop primer annealson a loop different from the loops annealed by the FIP and BIP primers,becoming an additional synthetic initial point to accelerateamplification.

Because it is advantageous to use the single-stranded loop region in thereaction of hybridizing a LAMP amplification product with a probenucleic acid, the detection region is placed in the loop region.

FIG. 5 is a schematic chart showing four kinds of single-stranded loopregions formed from the LAMP amplification product. The loop regionshave respectively the FP or BPc regions or the regions complementarythereto as detection regions, and the figure also shows probe nucleicacids hybridizing with the detection sequences. As shown in FIG. 5, thefour kinds of probe nucleic acids may be used for detection of theamplification products, but just one or two kinds of the probe nucleicacids may be used suitably.

In a first aspect of the present invention, the FP and F2 regions aredesigned to have an unoverlapping region of at least 10 bases or moreand overlapping regions of 10 bases or less. Similarly, the BPc and B2cregions are designed to have an unoverlapping region of at least 10bases or more and overlapping regions of 10 bases or less. Preferably,the overlapping region has a length of zero to 10 bases, more preferablyzero to 5 bases. As these regions are designed to satisfy the conditionsabove, other regions are also designed suitably, and the primers aredesigned properly according to the regions.

Primer design according to the conditions, with respective regionssatisfying the condition above, is important for improving thehybridization efficiency between the amplification products and theprobe nucleic acids. The reason will be described below in detail withreference to FIG. 6.

FIG. 6 is a schematic chart showing the FP region and the F2 regionlargely overlapping each other. FIG. 6A shows a case where the FP regionis located in the loop structure, i.e., when it is a plus chain. Theprobe nucleic acid at this time has a sequence complementary to that ofthe FP region (i.e., sequence of the FPc region). There is still theunreacted FIP primer in the reaction solution, which has the samesequence as that of the F2 region. As a result, as shown in FIG. 6A, theunreacted FIP primer and the probe nucleic acid hybridize with eachother with the sequence of the overlapping FP and F2 regions.Accordingly, the unreacted FIP primer, which functions as a competitivesubstance to the amplification product, leads to deterioration of thehybridization efficiency between the amplification products and theprobe nucleic acids. In addition, the unreacted primer may give rise toa pseudopositive signal by hybridization thereof with the probe nucleicacid, even when the target nucleic acid is not amplified.

FIG. 6B shows a case where the FPc region is located in the loopstructure, i.e., when it is a minus chain. As shown in FIG. 2, thenucleic acid chain has the F2c region complementary to the F2 region.Because the F2c region is complementary to the F2 region in FIP primer,the unreacted FIP primer forms a double-stranded chain by annealing onthe F2c region, leading to inhibition of the hybridization between theamplification product and the probe nucleic acid. The influence isfatal, particularly during real-time detection or high-sensitivityhigh-accuracy or detection of amplification products.

In summary, the hybridization inhibition factors described above may bedivided into the following two groups: (I) reactions between anunreacted primer and a probe nucleic acid, and (II) reactions between anunreacted primer and an amplification product. These inhibition factorsseem to cause problems such as deterioration of hybridization reactionefficiency and detection accuracy.

Hereinafter, a second aspect of the present invention where a loopprimer is used will be described. In such a case, the FP and LF regionsare designed to have an unoverlapping region of at least 10 bases ormore and overlapping regions of 10 bases or less. Similarly, the BPc andLBc regions are designed to have an unoverlapping region of at least 10bases or more and overlapping regions of 10 bases or less. As theseregions are designed to satisfy the conditions above, other regions arealso designed suitably, and the primers are designed properly accordingto the regions designed.

Also in the second aspect, significant overlapping between the FP regionand the loop primer region may lead to deterioration of hybridizationefficiency, as with the first aspect. For example as shown in FIG. 7A,in the case of a plus chain, the unreacted loop primer LFc forms adouble-stranded chain by annealing on the LF region in the amplificationproduct and inhibits hybridization with the probe nucleic acid FPc.Thus, the reaction is influenced by the inhibition factor (II). Also inthe case of a minus chain, the unreacted loop primer LFc hybridizes withthe probe nucleic acid FP, inhibiting hybridization between theamplification product and the probe nucleic acid, as shown in FIG. 7B.The unreacted loop primer may hybridize with the probe nucleic acid evenwhen the target nucleic acid is not amplified, giving rise to apseudopositive signal. Thus, the reaction is influenced by theinhibition factor (I).

In FIG. 7, the F2, FP, and LF regions are placed in this order, but maybe placed in the order of F2, LF, and FP regions.

FIG. 8 shows a list of the inhibition factors. Relationships when theunreacted primer gives a pseudopositive signal with respect to the probenucleic acid (inhibition factor I) are shown as shaded in FIG. 8A. Theinhibition factor (I) is generated, when the sequences of the targetnucleotides in the amplification product, and the sequences in the innerprimers (FIP and BIP primers) or the sequences of loop primers (LFc andLBc primers) are the same chain (i.e., they are both plus chains orminus chains). In other words, the sequence of the probe nucleic acidand the sequence of the inner or loop primer are reverse to each other.

FIG. 8B shows relationships where the unreacted primer competes with theprobe nucleic acid (inhibition factor II) as they are shaded. Theinhibition factor (II) is generated, when the sequences of the targetnucleic acids in the amplification product and the sequences of theinner or loop primers are reverse to each other. In other words, thesequence of the probe nucleic acid and the sequence of the inner or loopprimer are same chain.

As described above, when any two of the FP, F2, and LF regions or theBP, B2, and LBc regions are overlapping each other, the overlappingregion affects or inhibits hybridization and reduces its efficiency, ifthe overlapping region is excessively large. Noticing the problem aboveand after intensive studies, the inventors have found that it waspossible to eliminate such influences or reduce them into an allowablerange by shortening the overlapping region to 10 bases or less, as shownin the Examples below.

The two regions having an overlapping region of 10 bases or less maybecome vulnerable to the influences, if the base number of the otherunoverlapping regions is too small, and thus, the unoverlapping regionspreferably have a sequence of 10 bases or more. The length of theunoverlapping region is not particularly limited, as long as it isincorporated in the single-stranded loop-structured region, and may bedetermined arbitrarily according to the sequence of the target nucleicacid and the relationship between the sequence of each primer and thedetection sequence.

FIG. 9 shows design patterns of detection sequence regions (FP, FPc, BP,and BPc), inner primer sequence regions (F1c+F2 and B1c+B2), and loopprimer sequence regions (LFc and LBc) of each of the plus and minuschains of the target nucleic acid chain. FIGS. 9(1) and 9(2) show caseswhere both the detection sequence and the loop primer are located in theF region side. In FIG. 9(1), the FP region and the F2 or LF region aredesigned to have an unoverlapping region of at least 10 bases or moreand overlapping regions of 10 bases or less.

The reason for making the overlapping region between the FP region andthe F2 or LF region 10 bases or less is already described. For example,when the detection sequence is a plus chain (FP), the F2 region maycause the inhibition factor I, while the LFc region may cause theinhibition factor II. When the detection sequence is a minus chain(FPc), the LFc region may cause the inhibition factor I, while the F2region may cause the inhibition factor II.

The overlapping region between the LF and F1 regions is 20 bases orless, more preferably 10 bases or less in length. It is because the loopprimer LFc should anneal on the double-stranded chain in the F1-F1cregion, leading to decrease in amplification efficiency, when the basenumber in the overlapping region between the LF and F1 regions isincreased.

In the case of FIG. 9(2), the FP and LF regions are designed to have anunoverlapping region of at least 10 bases or more and overlappingregions of 10 bases or less. The reason for making the overlappingregion between the FP and LF regions 10 bases or less is alreadydescribed. For example, when the detection sequence is a plus chain(FP), the LFc region causes the inhibition factor II. When the detectionsequence is a minus chain (FPc), the LFc causes the inhibition factor I.

The number of bases in the duplicated region of the FP and F1 region ispreferably less than 15, more preferably zero. When the overlappingregion between the FP and F1 regions is 15 bases or more in length, theF1c region causes the inhibition factor I, when the minus chain (FPc) isdetected. It is because the probe nucleic acid should hybridize with thedouble-stranded F1-F1c region, leading to decrease in hybridizationefficiency.

The base number of the overlapping region between the F2 and LF regionsis preferably 20 or less, more preferably 10 or less. When the F2 and LFregions overlap each other over a length of 20 bases, the sequences ofthe FIP primer containing the F2 sequence and the loop primer LFc arecomplementary to each other in the overlapping region, resulting ininteraction between the primers. As a result, the primers become lessreactive with the amplification product on which it anneals, leading todecrease in amplification efficiency.

FIG. 9(3) shows a case where there is no loop primer in the F regionside. For the same reason as in FIGS. 9(1) and 9(2), the FP and F2regions are designed to have an unoverlapping region of at least 10bases or more and overlapping regions of 10 bases or less. The FP and F1regions are also designed to have an overlapping region of less than 15bases, more preferably zero base in length.

FIG. 9(4) shows a case where there is no detection sequence placed inthe F region side. The case 9(4) may be combined with the cases 9(6) to9(8) where the detection sequences are placed in the B region. For thesame reason as in FIGS. 9(1) and 9(2), the F2, LF, and F1 regions aredesigned to have an unoverlapping region of 20 bases or less and morepreferably 10 bases or less.

FIG. 9(5) shows a case where there is no loop primer or detectionsequence placed in the F region side. The case 9(5) may be combined withthe cases 9(6) to 9(8) where the detection sequences are placed in the Bregion. The F2 and F1 regions are preferably separated from each otherby 5 bases or more for improvement of self-annealing efficiency.

FIGS. 9(6) to 9(10) showing the B region are similar to the casesshowing the F region side. The cases 9(9) and 9(10) where there is nodetection sequence, may be combined with the cases 9(1) to 9(3) wheredetection sequences are placed in the F region side.

As shown in FIGS. 9(1), 9(2), 9(6) and 9(7), presence of a detectionsequence region and a loop primer sequence region in the same loopoccasionally results in deterioration of self-annealing efficiency andalso in amplification efficiency, by elongation of the chain in thesingle-stranded loop region, depending on the sequence of the targetnucleic acid. In such a case, it is effective to place a loop primer notin the loop region containing a detection sequence, but in another loopregion.

In a third aspect, the present invention provides a method of designinga primer used in the method of detecting single-nucleotide mutation(single-nucleotide polymorphism) by the LAMP method. The amplificationmethod and the detection method by using a probe nucleic acid by theLAMP method are the same as those in the first and second aspects, butthe site of single-nucleotide mutation in the target nucleic acid isdesigned to be included in the region of FP or BP, for detection of thesingle-nucleotide mutation. Thus, F3, F2 and F1 regions are placed inthis order from the 5′ terminal side of a target nucleic acid, B3c, B2cand B1c regions are placed in this order from the 3′ terminal side, andadditionally, an FP region containing single-nucleotide mutation isplaced in the region from the F2 to F1 regions above. Alternatively, theBPc region containing single-nucleotide mutation may be located in theregion from the B2c to B1c regions.

The primer used in the present aspect is the same as that used in thefirst aspect. In the third aspect, the F2 region is designed not tooverlap the single-nucleotide mutation site in the FP region.Alternatively, the B2c region is designed not to overlap thesingle-nucleotide mutation site in the BPc region. As these regions aredesigned to satisfy the conditions above, other regions are alsodesigned suitably, and the primers are designed properly according tothe regions designed.

For detection of single-nucleotide mutation, the amplification productmay be hybridized after it is amplified completely with a probe nucleicacid. Hybridization with the probe nucleic acid then is determined bythe base at the single-nucleotide mutation site in the amplificationproduct. In this method, reaction condition should be restricted owingto detect single-nucleotide mutation. Therefore, even if one shouldforget addition of a genome and an amplification reaction does not occurbut a primer exists so much in solution, a probe and a primer does nothybridized each other. So they are not determined incorrectly. Therestriction in primer design is thus alleviated, whether the unreactedprimers remains among the amplification product, because only thedifference in relative intensity of the signal associated withsingle-nucleotide mutation is to be differentiated.

However, design of an inner or loop primer at the single-nucleotidemutation site or the site of its complementary chain results inpreferential amplification of the genome with the genotype contained inthe inner or loop primer, prohibiting production of the amplificationproduct of the genotype inherent to the target nucleic acid.

It is thus important not to place an inner or loop primer sequenceregion at a position at the single-nucleotide mutation(single-nucleotide polymorphism) site in the target nucleic acid. Theregions are determined according to the setting above and the primer isdesigned.

In a fourth aspect, the present invention provides a method of designinga primer used in the method of detecting single-nucleotide mutation byusing a loop primer, as in the second aspect.

Thus, the LF region is placed in the region from the F2 to F1 regions,and the LBc region is placed in the region from the B2c to B1c regions.In addition to the primers above, the loop primer LFc consisting of thesequence complementary those of the LF region and the loop primer LBcconsisting of the sequence identical to the LBc region are used incombination. In the fourth aspect of the present invention, the LFcregion is placed at a position not overlapping the single-nucleotidemutation site in the FP region. Alternatively, the LBc region is placedat a position not overlapping the single-nucleotide mutation site in theBPc region. As these regions are designed to satisfy the conditionsabove, other regions are also designed suitably, and the primers aredesigned properly according to the regions designed.

FIG. 10 shows the design pattern of respective regions in the plus orminus chain of the target nucleic acid, when single-nucleotide mutationis to be detected. In FIGS. 10(1) and 10(2), both a detection sequenceand a loop primer region sequence are placed in the F region side. Inthe case of FIG. 10(1), the F2 or LF region or its complementary chainsite is placed at a position not overlapping the single-nucleotidemutation site contained in the FP or FPc region. Thus, the F2 region mayoverlap the FP region to the base adjacent to the mutation site.Similarly, the LF region may overlap the FPc region to the base adjacentto the mutation site.

The LF and F1 regions preferably have an overlapping region of 20 basesor less, more preferably an overlapping region of 10 bases or less. Agreater number of bases overlapped in the LF and F1 regions isundesirable, because it demands annealing of the loop primer LFc on thedouble-stranded F1-F1c region, leading to deterioration of amplificationefficiency.

In FIG. 10(2), the single-nucleotide mutation site contained in the FPregion is placed as separated from the LF region. In other words, the LFregion may overlap the FP region to the base adjacent to the mutationsite. The overlapping region between the FP region and the F1 region ispreferably less than 15 bases, more preferably zero bases, in length.The base number is preferably in the above range, because a greater basenumber of the FP and F1 overlapping region demands hybridization of theprobe with the double-stranded F1-F1c region, leading to deteriorationof hybridization efficiency.

The base number of the F2 and LF overlapping region is preferably 20bases or less, more preferably 10 bases or less. A greater base numberof the F2 and LF overlapping region leads to expansion of the sequenceregions where the F2 sequence in the inner primer and the loop-primerFPc sequence are complementary to each other and to interaction betweenthe primers, and consequently to deterioration of reactivity with thetarget nucleic acid on which it anneals and in amplification efficiency.Accordingly, the base number is preferably in the range above.

In FIG. 10(3), there is no loop primer placed in the F region side. Aswith FIGS. 10(1) and 10(2), the F2 region may overlap the FP region tothe base adjacent to the mutation site, and the number of bases in theoverlapping region between the FP and F1 regions is preferably less than15, more preferably zero.

In FIG. 10(4), there is no detection sequence placed in the F regionside. The case of FIG. 10(4) may be combined with the case shown inFIGS. 10(6) to (8) in which there is a detection sequence in the Bregion. As with the cases of FIGS. 10(1) and (2), the numbers of theoverlapping regions between the F2 and LF regions and the LF and F1regions are preferably 20 or less, more preferably 10 or less.

In FIG. 10(5), there is no loop primer or detection sequence placed inthe F region side. The case of FIG. 10(5) may be combined with the caseshown in FIGS. 10(6) to (8) in which there is a detection sequence inthe B region. The F2 and F1 regions may be separated by 5 bases or morefor improvement in self-annealing efficiency.

FIGS. 10(6) to (10) show the cases of the B region, which are the sameas the cases of the F region side. The case shown in FIG. 10(9) or (10)in which there is no detection sequence may be combined with the caseshown in FIGS. 10(1) to (3) in which there is a detection sequence inthe F region.

In detecting single-nucleotide mutation, if the detection sequenceregion or the sequence region of its complementary chain, and the loopprimer sequence region are placed in the same loop, the single-strandedloop region becomes a long chain lower in self-annealing efficiency,leading to deterioration of amplification efficiency, depending on thesequence of the target nucleic acid. In such a case, it is effective touse a method of shortening the loop region by eliminating the loopprimer in the detection sequence-containing loop and place the loopprimer in the other loop containing no detection sequence.

In a fifth aspect, the present invention provides a detection method ofamplifying a target nucleic acid by the LAMP method and detecting itwith a probe nucleic acid, or a method of amplifying a target nucleicacid containing single-nucleotide mutation by the LAMP method anddetecting the single-nucleotide mutation with a probe nucleic acid. Inthe fifth aspect, the target nucleic acid is amplified by the LAMPmethod by using a primer designed in the first to fourth aspects and theamplification product is detected by hybridization with a probe nucleicacid.

The probe nucleic acid for use has a sequence complementary to thedetection sequence (FP, FPc, BP, or BPc), as described above. The probenucleic acid may be prepared for any one of the detection sequences orfor multiple detection sequences. The probe nucleic acid may be presentas dissolved in solution, or alternatively, may be immobilized, forexample, on an aggregation carrier or a solid-phase support.

The probe nucleic acid may be unlabeled or labeled with a fluorescentmaterial such as Cy5, Cy3, FITC or rhodamine, a light-emitting substancesuch as luminol, lucigenin or an acridinium ester derivative, hapten oran enzyme, according to the detection method. Yet alternatively, theprobe nucleic acid may be modified with a reactive functional group suchas amino, carboxyl, hydroxyl, thiol, or sulfone group or with asubstance such as avidin or biotin, for immobilization of the probenucleic acid on the support.

Examples of the supports on which the probe nucleic acid is immobilizedinclude nitrocellulose film, nylon film, microtiter plate, glass,electrode, magnet, beads, plastics, latex, synthetic resins, naturalresins, optical fiber, and the like.

Alternatively, a DNA chip may be formed with the probe nucleic acid. TheDNA chip is a device having a substrate such as glass or silicon andprobe nucleic acids immobilized thereon at high density that allowsacquisition of much gene sequence information. The current mainstreamfluorescent detection method is a method of detecting afluorescent-labeled sample gene with a high-sensitivity fluorescenceanalyzer after allowing it to react with a probe on chip. Also underdevelopment as an alternative detection method is an electriccurrent-detecting DNA chip. It is a method of hybridizing a probenucleic acid immobilized on an electrode with a target nucleic acid,adding an intercalating agent that reacts with double-stranded chainDNAs specifically, and measuring the electrochemical signal obtainedfrom the intercalating agent. The electrochemical DNA chip, whichdemands no labeling or no expensive device for detection, is attractingattention as a second-generation DNA chip (see, e.g., JP-A 5-199898[KOKAI]).

When two or more probe nucleic acids are used, unlabeled and labeledprobes, or probes immobilized or unimmobilized on solid phase, may beused at the same time, according to the detection method. For example,in the sandwich hybridization using a probe nucleic acid for capturingand a labeled probe nucleic acid for detection, the labeled probenucleic acid is hybridized with the detection sequence as it isdissolved in a solution, but the probe nucleic acid for capturing may beimmobilized on a support after dissolved in solution and hybridized withthe detection sequence or may be immobilized previously on the support.

Hybridization and Reaction Condition

Hybridization between the amplification product and the probe nucleicacid is carried out under a suitable condition. The suitable conditionvaries according to the kind and structure of the amplification product,the kind of the bases contained in the detection sequence, and the kindof the probe nucleic acid. It is carried out, for example, in a buffersolution at an ionic strength in the range of 0.01 to 5 and a pH in therange of 5 to 10. Other additives, for example, a hybridizationaccelerator such as dextran sulfate, salmon sperm DNA, bovine thymicDNA, EDTA and a surfactant, may be added to reaction solution. Thereaction temperature is, for example, in the range of 10° C. to 90° C.,and the reaction may be accelerated by agitation or shaking. Afterreaction, the support may be washed, for example, with a buffer solutionat an ionic strength in the range of 0.01 to 5 and a pH in the range of5 to 10.

Detection Method

Any method may be used for detecting an amplification product hybridizedwith a probe nucleic acid. Typical examples thereof include thefollowings:

The first detection method is a method of using an intercalating agentbinding to nucleic acid double-stranded chains. In the method, the probenucleic acid is modified with a reactive functional group such as amino,carboxyl, hydroxyl, thiol, or sulfone group or with a substance such ashapten, avidin, or biotin, and immobilized on a solid phase. Theamplification product and the probe nucleic acid immobilized on a solidphase are hybridized with each other, and the resulting support is thenwashed. Hybridization between the probe nucleic acid and the amplifiedproduct is detected with a signal of the intercalating agent bound tothe double-stranded chains.

The intercalating agent is not particularly limited, as long as it isphotochemically or electrochemically active. Examples thereof includeethidium, ethidium bromide, acridine, aminoacridine, acridine orange,proflavin, ellipticine, actinomycin D, daunomycin, mitomycin C, Hoechst33342, Hoechst 33258, aclarubicin, DAPI, adriamycin, epirubicin,aclacinomycin, and the like. Other usable intercalating agents includethose described in JP-A 62-282599 (KOKAI).

In addition to substances described above reversible inoxidation/reduction reaction, metal complexes containing a substanceelectrically reversible in oxidation/reductive reaction as the centralmetal, i.e., metallointercalators, may be used as the intercalatingagents in detecting electrochemical change by using an electrode.Examples of the metallointercalators include tris(phenanthroline)zinccomplexes, tris(phenanthroline)ruthenium complexes,tris(phenanthroline)cobalt complexes, di(phenanthroline)zinc complexes,di(phenanthroline)ruthenium complexes, di(phenanthroline)cobaltcomplexes, bipyridine platinum complexes, terpyridine platinumcomplexes, phenanthroline platinum complexes, tris(bipyridyl)zinccomplexes, tris(bipyridyl)ruthenium complexes, tris(bipyridyl)cobaltcomplexes, di(bipyridyl)zinc complexes, di(bipyridyl)rutheniumcomplexes, and di(bipyridyl)cobalt complexes.

When performing gene detection by using an electrode, it is possible touse an intercalating agent showing electrochemical light emission. Suchan intercalating agent is not particularly limited, and examples thereofinclude luminol, lucigenin, pyrene, diphenylanthracene, and rubrene. Theelectrochemical light emission by these intercalating agents can beamplified with an enhancer, for example, a luciferin derivative such asfirefly luciferin or dehydroluciferin, phenols such as phenylphenol orchlorophenol, or naphthols.

When an optically active intercalating agent is used, the difference insignal between when the intercalating agent is alone and when it isbound to a double-stranded chain is detected, based on opticalinformation, such as absorbance, fluorescence, light emission, lightquenching, fluorescent polarization, or circular dichroism, or on thechange in absorption wavelength, fluorescent wavelength, emissionwavelength, or quenched light wavelength of the intercalating agentbound to a double-stranded chain. When an electrochemically activeintercalating agent is used, the oxidation/reduction current of thecentral metal or the intercalating agent itself is measured fordetection.

In the second detection method, a method of sandwich-hybridizing acapturing probe nucleic acid with a labeled probe nucleic acid fordetection can be used. The probe nucleic acid for capturing isimmobilized on a solid phase according to the method described in thefirst detection method. The substance labeling the nucleic acid to bedetected may be determined according to the subsequent detection methodused. Examples thereof include electrode active materials, fluorescentmaterials, light-emitting substances, electrochemical light-emittingsubstances, enzymes, enzyme substrates, hapten, antigens, antibodies,radio isotopes, and the like. When a substance prohibiting direct signaldetection such as hapten is used, the gene is detected indirectly byusing an enzyme-bound anti-hapten antibody such as enzyme-bound avidin,by measuring the optical information, such as turbidity, absorbance,fluorescence, light emission, light quenching, fluorescent polarization,or circular dichroism, or the electrical activity of the substanceduring enzyme reaction.

Alternatively when the DNA chip is made of a probe nucleic acid, thegene may be detected, for example, by a fluorescence-detecting method ora current-detecting method.

When using the current-detecting method with an intercalating agentbinding to nucleic acid double-stranded chains, the analyte substance isdetected generally according to the first detection method, placingelectrodes on a substrate of glass or silicon and immobilizing a probenucleic acid on the electrodes. The number and placement of theelectrodes can be determined easily as needed by those who are skilledin the art. A counter electrode and a reference electrode may also beused additionally, as in other common electrochemical detection methods.

The electrode for use is not particularly limited, and examples of thematerials for the electrode include pure metals such as gold, silver,platinum, mercury, nickel, palladium, silicon, germanium, gallium, andtungsten and the alloys thereof; carbons such as graphite and glassycarbon; and the oxides and compounds thereof.

Subsequently, the target nucleic acid is then allowed to hybridize withthe probe nucleic acid immobilized on the electrode and then, anelectrochemically active intercalating agent is added. Hybridizationbetween the target nucleic acid and the probe nucleic acid on thesubstrate is detected with the electrochemical signal from theintercalating agent.

The electrochemical measurement is performed, for example, by applying avoltage higher than that causing electrochemical reaction of theintercalating agent and measuring the reaction current derived from theintercalating agent. The voltage may be altered linearly, or may beapplied in the pulse shape or at a constant voltage. The current orvoltage during measurement may be controlled by using a device such aspotentiostat, digital multimeter, or function generator.

Examples of the intercalating agents entering the double-stranded chaininclude electrochemically active substances such as Hoechst 33258,acridine orange, quinacrine, daunomycin, metallointercalators,bisintercalators such as bisacridine, trisintercalators,polyintercalators, and the like. In addition, the intercalator may bemodified with an electrochemically active metal complex such asferrocene or viologen.

In the case of the fluorescence detection method, used is a method oflabeling the probe nucleic acid with a fluorescence colorant such asCy5, Cy3, FITC, or rhodamine, and sandwich-hybridizing the targetnucleic acid according to the second detection method, or a method oflabeling the target nucleic acid, for example, with a primer or dNTPlabeled with a fluorescence colorant and then hybridizing it with aprobe nucleic acid immobilized on a solid phase. The target nucleic acidbound to the probe nucleic acid on the solid phase is detected with asuitable detecting device properly selected according to the kind of thelabel used.

Analyte Sample

The sample to be analyzed in the present invention is not particularlylimited, and examples thereof include blood, serum, leukocyte, urine,feces, semen, saliva, tissue, biopsy sample, oral mucosa, cultured cell,sputum, and the like collected from individuals. The individual may behuman, animal other than human, plant, or microbial organism such asvirus, microbe, bacteria, yeast or mycoplasma. Nucleic acid componentsare extracted from these analyte samples, for preparation of a samplesolution used in the target nucleic acid detection test. The extractionmethod is not particularly limited, and examples thereof includecommercially available nucleic acid extraction kits such as QIAamp(manufactured by QIAGEN) and Sumaitest (manufactured by Sumitomo MetalIndustries, Ltd.), and the like.

Primer

Nucleic acid synthesis from the inner primer should be initiated beforesynthesis from the outer primer in the LAMP reaction, and thus, themelting temperature (Tm) of the inner primer should be higher than thatof the outer primer and/or the amount of the inner primer greater thanthat of the outer primer for efficient reaction. More specifically, themelting temperature (Tm) preferably satisfies the following formula:(melting temperature of F3-F3c or B3-B3c regions)≦(melting temperatureof F2-F2c or B2-B2c regions)≦(melting temperature of F1-F1c or B1-B1cregions). The formula (melting temperature of F2-F2c or B2-B2cregions)≦(melting temperature of F1-F1c or B1-B1c regions) is ensured tomake intramolecular annealing between F1 and F1c and between B1 and B1cproceed preferentially before annealing of F2 or B2 on the loop region.As for the ratio of the primers, the concentration of the inner primeris 2 to 50 times, preferably 4 to 25 times, larger than that of theouter primer.

Primer Length

The chain length of the six kinds of primers used in the LAMP reactionis preferably 10 to 100 bases. The inner primer has two regions bound toeach other, and the chain length of the inner primer is that of eachregion. The length of 10 bases or more is a length needed for annealingof the primer on a template, while specificity is preserved. As it isdifficult to prepare an excessively lengthy base by chemical synthesis,the chain length is preferably in the range above.

The distance from the F2c region to the F1c region and from the B2cregion to the B1c region which is the single-stranded loop region, i.e.,the base number, is preferably zero to 120 bases, more preferably 5 to70 bases. The regions F2c and B2c are not included in the value. Thechain length above is desirable, because an excessively longer orshorter single-stranded loop region results in inefficientself-annealing.

The base number of the chain between the F1 and B1 regions (excluding F1and B1 regions) may be zero, but is preferably 10 or more. Ashorter-length chain between the regions F1 and B1 may lead tounstabilized amplification. The base number of the chain between theregions F2 and B2 (including F2 and B2 regions) is preferably 700 orless, more preferably 500 or less, although it depends on the activityof the strand-displacing polymerase used.

DNA Polymerase

Examples of the strand-displacing DNA polymerases include Bst DNApolymerase, Bca(exo-)DNA polymerase, Vent DNA polymerase, Vent(exo-)DNApolymerase, DeepVent DNA polymerase, DeepVent(exo-)DNA polymerase, andthe like, as well as the DNA polymerases described in Japanese PatentNo. 3313358.

LAMP Reaction Condition

The temperature and pH of the LAMP reaction are adjusted dependent onthe DNA polymerase enzyme to give it a favorable activity. In addition,a salt may be appropriately added for adjustment of the enzyme activityor the melting temperature (Tm) of the nucleic acid. Examples thereofinclude KCl, NaCl, (NH₄)₂SO₄, and the like. In addition, adjustmentagent of melting temperature (Tm) such as betaine, DMSO, or the like maybe used. Also, enzyme protectant such as bovine serum albumin orsaccharide, or the like may be used. See JP 3313358 for details.

EXAMPLES

Hereinafter, the present invention will be described specifically withreference to Examples.

Example 1 Detection of Pseudopositive Signal Caused by Inner PrimerDetection Sequence

In the present Example, human MTHFR gene was used as the sample nucleicacid. The detection sequence was placed in the single-stranded loopregion of the MTHFR LAMP product, either with (i) 0, (ii) 5, (iii) 10,or (iv) 15 bases overlapping the F2 sequence of FIP primer (FIG. 11).

(i) AGGAGCTGACCAGTGAAGAAAGTG (ii) GGGGGAGGAGCTGACCAG (iii)ATGTGGGGGGAGCAGCTG (iv) TGAAGATGTGGGGGGAGG

Primer

The synthetic nucleotide primers used in the present Example are listedbelow. FIG. 11 shows the sequence region of each primer in the humanMTHFR gene. The FIP primer contains F1c and F2 regions, and the BIPprimer contains B1c and B2 regions. Any sequence may or may not beinserted between them. In FIG. 11, a reverse chain having a sequencecomplementary to the sequence shown in FIG. 11 was used as the c-addedregion (i.e., F1c) in the F region, while a reverse chain having thesequence shown in FIG. 11 as the non-c-added regions (i.e., B2 and B3)in the B region.

MTHFR F3 primer GCTGAAGGACTACTACCTCTTCTACC MTHFR FIP primerCGGTTTGGTTCTCCCGAGAG(F1c)-GCTGCTGAAGATGTGGGGGG(F2) MTHFR B3 primerGCACAGGATGGGGAAGTC MTHFR BIP primerGTGAGTGATGCTGGAGTGGG(B1c)-AGCTGGGGTCAGGCC(B2)

LAMP Reaction Solution

A solution for LAMP reaction was prepared according to the following.The LAMP reaction performed with a human or mouse genome as thetemplate. A LAMP reaction solution was allowed to hybridize with a probenucleic acid immobilized on a gold electrode. Then, the detectionsequence present in the LAMP amplification product was detected by usinga current-detecting method.

The LAMP reaction solution comprising:

Sterilized ultrapure water 5.5 μLBst DNA polymerase 1 μL

Buffer 12.5 μL Tris.HCl pH 8.0 40 mM KCl 20 mM MgSO₄ 16 mM (NH₄)₂SO₄ 20mM Tween 20 0.2% Betaine 1.6 M DNTP 2.8 mM

F3-primer (10 μM) 0.5 μLB3-primer (10 μM) 0.5 μL

FIP-primer (20 μM) 2 μL BIP-primer (20 μM) 2 μL

30 ng/μl template (purified human or mouse genome) 1 μLTotal amount 25 μL

Amplification Reaction by LAMP Method

A sample was amplified by using human or mouse genomes, or by usingsterile ultrapure water replacing the template as control, in the LAMPreaction solution having the composition above at 63° C. for 120minutes. Agarose electrophoresis of the amplification product showedbands associated with amplification in the product amplified with thehuman genome template, but no amplification band in the productamplified with the mouse genome and sterile ultrapure water replacingthe template. There was white precipitate observed in the solution ofthe product amplified with the human genome template afteramplification, but there was no while precipitate in the solutions ofthe product amplified with the mouse genome template and the productamplified with the sterile ultrapure water. The white precipitation is aphenomenon observed in the solution caused by a byproduct formed in theamplification process, magnesium pyrophosphate, during LAMPamplification.

These results indicated that the product amplified with the human genometemplate was formed in specific amplification reaction, while noamplification reaction occurred when the sample was amplified with themouse genome template or sterile ultrapure water replacing the template.

Preparation of Probe Nucleic Acid-Immobilized Electrode

The nucleotide sequences of the probe nucleic acids used are shownbelow.

Negative probe GACTATAAACATGCTTTCCGTGGCA Positive probe (i)CACTTTCTTCACTGGTCAGCTCCT Positive probe (ii) CTGGTCAGCTCCTCCCCC Positiveprobe (iii) CAGCTCCTCCCCCCACAT Positive probe (iv) CCTCCCCCCACATCTTCA

The positive probes (i) (ii) (iii) and (iv) were minus chain modified5′-terminal with SH-group. The negative probe had a sequence unrelatedto the MFHFR gene sequence and modified 5′-terminal with SH-group.

Each probe was immobilized on a gold electrode, with the strong chemicalbonding potential between a thiol group and gold. A solution containingprobe was spotted on the gold electrode and left at 25° C. for 1 hour,and then, the electrode was immersed in 1-mM mercaptohexanol solutionand washed with 0.2×SSC solution. The same probe solution was spotted onthree electrodes. After washing, the electrode was washed with ultrapurewater, and dried in air, to give a probe-immobilized electrodesubstrate.

Electrode Allocation

Electrodes 1 to 3: negative probe

Electrodes 4 to 6: positive probe (i)

Electrodes 7 to 9: positive probe (ii)

Electrodes 10 to 12: positive probe (iii)

Electrodes 13 to 15: positive probe (iv)

Preparation of Reaction Solution and Hybridization

The reaction solution comprised the product amplified with the human ormouse genome or sterile ultrapure water as template respectively, andadded a salt diluted to a final concentration of 2×SSC. Separately, asolution at a final concentration of 2×SSC was also prepared as thecontrol. Each of these solutions was spotted on the probe nucleicacid-immobilized electrode substrate prepared above and left at 35° C.for 60 minutes, allowing progress of hybridization reaction. Then, theelectrodes were cleaned with ultrapure water slightly. The electrode wasimmersed in a phosphate buffer containing 50 μM of an intercalatingagent Hoechst 33258 for 15 minutes, and the oxidation current responseof the Hoechst 33258 molecule was measured.

Results

As shown in FIG. 12, the control 2×SSC solution indicated no increase insignal intensity on the positive probe (i) to (iv)-immobilizedelectrodes, compared to the current observed on the negative probe(NP)-immobilized electrode. On the other hand, with the productamplified with the human genome template, observed were signalsassociated with hybridization on all the positive probe (i) to(iv)-immobilized electrodes.

Although there was no amplification reaction occurring with the productamplified with the mouse genome template and the product amplified withsterile ultrapure water replacing the template, a pseudopositive signalof an oligonucleotide having 15 bases overlapping the F2 region wasobserved from the positive probe (iv). In addition, the pseudopositivesignal disappeared, as the number of overlapping bases in the positiveprobe was reduced, i.e., when the positive probe was changed frompositive probe having ten overlapping bases (iii) and positive probehaving five overlapping bases (ii) to positive probe having nooverlapping base (i).

These results indicated that overlapping of the detection sequence andthe inner primer sequence in the single-stranded loop gave rise to apseudopositive signal. Seemingly, it is because the probe nucleic acidhaving a sequence complementary to the detection sequence hybridizeswith the inner primer. Obviously, presence of an overlapping region of15 bases results in a distinct pseudopositive signal, prohibitingaccurate detection results.

Example 2 Detection of Pseudopositive Signal Caused by Loop PrimerDetection Sequence

In the present Example, human MTHFR gene was used as sample nucleicacid, as in Example 1. The detection sequence was placed in thesingle-stranded loop region of the MTHFR LAMP product. The detectionsequences used were four kinds of detection sequences respectivelyhaving (v) no bases, (vi) 5 bases, (vii) 10 bases, and (viii) 15 basesoverlapping the sequence of the loop primer LFc (FIG. 13).

(v) TTTCTTCACTGGTCAGCTCCT (vi) GACACTTTCTTCACTGGTCAG (vii)TCAAAGACACTTTCTTCACTG (viii) AGACTTCAAAGACACTTTCTT

1) Primer

The synthetic nucleotide primers used in the present Example are shownbelow. FIG. 13 shows the sequence region of each primer in human MTHFRgene. The FIP primer has F1c and F2 regions, while the BIP primer hasB1c and B2 regions. The sequence regions were expressed similarly toExample 1.

MTHFR F3 primer GCTGAAGGACTACTACCTCTTCTACC MTHFR FIP primerCGGTTTGGTTCTCCCGAGAG(F1c)-GCTGCTGAAGATGTGGGGGG(F2) MTHFR B3 primerGCACAGGATGGGGAAGTC MTHFR BIP primerGTGAGTGATGCTGGAGTGGG(B1c)-AGCTGGGGTCAGGCC(B2) MTHFR LFc primerGTAAAGAACAAAGACTTCAAAGACAC MTHFR LBc primer CCCTGGTTCATCCCCTG

2) LAMP Reaction Solution

A solution for LAMP reaction was prepared according to the following.The LAMP reaction performed with a human or mouse genome as thetemplate. A LAMP reaction solution was allowed to hybridize with a probenucleic acid immobilized on a gold electrode. Then, the detectionsequence present in the LAMP amplification product was detected by usinga current-detecting method.

The LAMP reaction solution comprising:

Sterile ultrapure water 1.5 μLBst DNA polymerase 1 μL

Buffer 12.5 μL Tris.HCl pH 8.0 40 mM KCl 20 mM MgSO₄ 16 mM (NH₄)₂SO₄ 20mM Tween 20 0.2% Betaine 1.6 M DNTP 2.8 mM

F3-primer (10 μm) 0.5 μLB3-primer (10 μm) 0.5 μL

FIP-primer (20 μm) 2 μL BIP-primer (20 μm) 2 μL

LFc primer (10 μm) 2 μLLBC primer (10 μm) 2 μL30 ng/μl template (purified human or mouse genome) 1 μLTotal amount 25 μL

Amplification Reaction by LAMP Method

A sample was amplified by using human or mouse genomes, or by usingsterile ultrapure water replacing the template as control. In FIG. 14A,four kinds of basic primers (F3, B3, FIP, and BIP primers) and two kindsof loop primers (LFc and LBc primers) were used in the compositiondescribed in 2) above. In FIG. 14B, five kinds of primers out of the sixkinds of primers above (excluding LFc primer) were added in thecomposition above, and sterilized water was added instead of the LFcprimer. In FIG. 14C, four kinds of primers out of the six kinds ofprimers (excluding the LFc and LBc primers) were used in the compositiondescribed above, and sterilized water was added instead of the LFc andLBc primers.

The LAMP reaction was carried out at 63° C. for 30, 60, 90, or 120minutes. Agarose electrophoresis of the amplified product similar toExample 1 revealed that the product amplified with the human genometemplate gave amplification bands with all three kinds of primer sets,while the products amplified with the mouse genome template and withsterile ultrapure water replacing the template gave no amplificationband with any of the three kinds of primer sets. There was whiteprecipitate in the solution of the product amplified with the humangenome template after amplification, while there was no whiteprecipitate in the solutions of the product amplified with the mousegenome template and with sterile ultrapure water replacing the template.These results confirmed that a specific amplification reaction occurredwith the product amplified with the human genome template while noamplification reaction proceeded with the product amplified with themouse genome template or with sterile ultrapure water replacing thetemplate.

Preparation of Probe Nucleic Acid-Immobilized Electrode

The nucleotide sequences of the probe nucleic acids used are shownbelow.

negative probe GACTATAAACATGCTTTCCGTGGCA positive probe (v)AGGAGCTGACCAGTGAAGAAA positive probe (vi) CTGACCAGTGAAGAAAGTGTC positiveprobe (vii) CAGTGAAGAAAGTGTCTTTGA positive probe (viii)AAGAAAGTGTCTTTGAAGTCT

The positive probes (v) to (viii) were plus chain modified 5′-terminalwith SH-group. The negative probe had a sequence unrelated to the MTHFRgene sequence and modified 5′-terminal with SH-group. Each of the probeswas immobilized on a gold electrode, according to a method similar tothat in Example 1.

Electrode Allocation

Electrodes 1 to 3: negative probeElectrodes 4 to 6: positive probe (v)Electrodes 7 to 9: positive probe (vi)Electrodes 10 to 12: positive probe (vii)Electrodes 13 to 15: positive probe (viii)

Preparation of Reaction Solution and Hybridization

Three kinds of reaction solutions respectively containing a humangenome, a mouse genome, and sterile ultrapure water replacing the genomewere subjected to amplification by using three kinds of primer sets A, Band C described above as templates at 63° C. for 120 minutes. Afteramplification, a salt was added to each reaction solution to a finalconcentration of 2×SSC. A solution of 2×SSC was also prepared as thecontrol. Each of these solutions was spotted on the probe nucleicacid-immobilized electrode substrate prepared above and left at 35° C.for 60 minutes, allowing hybridization reaction. Then, electrochemicalmeasurement was performed according to a method similar to that inExample 1.

Results

As shown in FIG. 14, with the control 2×SSC solution, no increase insignal was observed from the positive probe (v), (vi), (vii), or(viii)-immobilized electrode, compared to the current from the negativeprobe-immobilized electrode. On the other hand, increase in the signalfrom the positive probes (v) (vi) (vii) and (viii) associated withhybridization was observed from the product amplified with the humangenome template.

With the product amplified with the mouse genome template and withsterile ultrapure water replacing the template, a pseudopositive signalwas detected from a positive probe (viii) having a detection sequencehaving 15 bases overlapping the sequence of loop primer LFc, although noamplification reaction occurred. The pseudopositive signal decreased, asthe number of overlapping bases in the positive probes is reduced, i.e.,when the positive probe is changed from positive probe having tenoverlapping bases (vii) and positive probe having five overlapping bases(vi) to positive probe having no overlapping base (v).

These result revealed that overlapping of the detection sequence and theloop primer sequence in the single-stranded loop leads to hybridizationof the probe nucleic acid having a sequence complementary to thedetection sequence with the loop primer in solution, giving apseudopositive signal.

As shown in FIG. 15, in the present Example, a pseudopositive signal wasdetected as described above in the case A where the analyte sample wasamplified with six kinds of primers including a loop primer LFc placedin the F region side having a region overlapping the detection sequence.Alternatively in the case C where the analyte sample was amplified withfour kinds of primers without addition of either loop primer LFc or LBc,the amplification efficiency was lower and it took almost 120 minutesfor complete amplification.

Yet alternatively in the case of B where the analyte sample wasamplified with five kinds of primers together with a primer having aloop primer LBc only in the B region where there is no detectionsequence, the pseudopositive was not detected and the amplification wascompleted in 60 minutes. These results indicated that, if it was notpossible to place a detection sequence or a sequence complementary tothe detection sequence and a loop primer in the same loop, a method ofplacing the loop primer in a loop different from the loop containing thedetection sequence or the sequence complementary to the detectionsequence was effective in terms of LAMP amplification efficiency anddetection efficiency of the amplification product.

Example 3 Inhibition of hybridization reaction Between Probe NucleicAcid and Detection Sequence by Loop Primer Detection Sequence

In the present Example, human MTHFR gene was used as the sample nucleicacid as in Example 2. The detection sequence was placed in thesingle-stranded loop region of the MTHFR LAMP product, and four kinds ofdetection sequences, sequences complementary to the detection sequencehaving (ix) no bases, (x) 5 bases, (xi) 10 bases, (xii) 15 basesoverlapping the sequence of the loop primer LFc, were prepared (FIG.13). The detection sequences used in Example 3 are reverse chains ofthose used in Example 2 (i.e., complementary sequences in the oppositedirection).

(ix) AGGAGCTGACCAGTGAAGAAA (x) CTGACCAGTGAAGAAAGTGTC (xi)CAGTGAAGAAAGTGTCTTTGA (xii) AAGAAAGTGTCTTTGAAGTCT

1) Primer

Primers similar to those used in Example 2 were used.

2) LAMP Reaction Solution

A LAMP reaction solution containing a human genome as the template wasallowed to hybridize with a probe nucleic acid immobilized on a goldelectrode. Then, the detection sequence present in the LAMPamplification product was detected by using a current-detecting method.The LAMP reaction solution has a composition similar to that in Example2.

Amplification Reaction by LAMP Method

A. a LAMP reaction solution containing six kinds of primers, B. a LAMPreaction solution containing five kinds of primers without the LFcprimer, and C. a LAMP reaction solution containing four kinds of primerswithout the LFc or LBc primer, respectively containing a human genomeadditionally as the template, were prepared. Each of these solutions wasallowed to react at 63° C. for 30, 60, 90, or 120 minutes. The amplifiedproduct was analyzed by agarose electrophoresis. In addition, theamplified product in a solution added with sterile ultrapure waterreplacing the genome during preparation of the LAMP reaction solutionwas also analyzed by electrophoresis at the same time, confirmingabsence of contamination.

Preparation of Probe Nucleic Acid-Immobilized Electrode

The nucleotide sequences of the probe nucleic acids used are shownbelow.

negative probe GACTATAAACATGCTTTCCGTGGCA positive probe (ix)TTTCTTCACTGGTCAGCTCCT positive probe (x) GACACTTTCTTCACTGGTCAG positiveprobe (xi) TCAAAGACACTTTCTTCACTG positive probe (xii)AGACTTCAAAGACACTTTCTT

The positive probes were minus chain modified 3′-terminal with SH-group.The negative probe had a sequence unrelated to the MTHFR gene sequenceand modified 3′-terminal with SH-group. The probe was immobilized on agold electrode according to a method similar to that in Example 1. Thesame probe solution was also spotted on three electrodes similarly.

Electrode Allocation

Electrodes 1 to 3: negative probeElectrodes 4 to 6: positive probe (ix)Electrodes 7 to 9: positive probe (x)Electrodes 10 to 12: positive probe (xi)Electrodes 13 to 15: positive probe (xii)

Dilution and Hybridization of LAMP Amplification Product

The reaction solution containing a human genome as the template wasamplified by using the primer sets A or B described above at 63° C. for30 or 60 minutes. Electrophoresis of the amplification product showedthat the primer sets A and B gave an amplification band afteramplification for 30 minutes thinner than that after amplification for60 minutes. The result confirmed that the product did not reachsaturated amplification after amplification for 30 minutes.

The amplification products in the cases of A and B after reaction for 60minutes and the amplification product in the case of A after reactionfor 30 minutes were diluted with LAMP buffer in a stepwise manner. Then,they were subjected to electrophoresis, together with the amplificationproduct in the case of B after reaction for 30 minutes; and thesolutions were diluted to a similar dilution ratio, to make theconcentration of these amplification products similar to each other.

Then, a salt was added respectively to the amplification product of Bafter reaction for 30 minutes, the amplification products of A and Bafter reaction for 60 minutes which was previously diluted with LAMPbuffer, and the amplification product of A after reaction for 30minutes, respectively to a final concentration of 2×SSC. A solution at afinal concentration of 2×SSC was also prepared as the control. Each ofthese solutions was spotted on the probe nucleic acid-immobilizedelectrode substrate prepared above and left at 35° C. for 60 minutes,allowing progress of hybridization reaction. Then, the electrode wascleaned with ultrapure water slightly. The electrode was immersed in aphosphate buffer containing 50 μM of an intercalating agent Hoechst33258 for 15 minutes, and the oxidation current response of the Hoechst33258 molecule was measured.

Results

As shown in FIG. 16, the amplification products of B after reaction for60 minutes and 30 minutes showed increase in signal almost at the samelevel from the electrodes having immobilized positive probes (ix) (x)(xi) and (xii). On the other hand, analysis of the amplificationproducts of A after reaction for 60 minutes and 30 minutes showed thatincrease in signal from positive probe (xii) having a detection sequencehaving 15 bases overlapping the sequence of the loop primer LFc waslower after 30 minutes than after 60 minutes. In addition, the increasein signal after reaction for 60 minutes and for 30 minutes became almostthe same, as the overlapping region is shortened, i.e., when thepositive probe was changed from the positive probe having tenoverlapping bases (xi) and the positive probe having five overlappingbases (x) to the positive probe having no overlapping base (ix).

The amplification product of A after reaction for 30 minutes is stillunder amplification reaction and contains the unreacted LFc primer in anamount greater than that of the amplification product of A afterreaction for 60 minutes. This indicates that in the solution containinga greater amount of residual unreacted LFc primer, the LFc primer in thesolution is annealed preferentially with the single-stranded detectionsequence faster than the probe immobilized on a solid phase, leading todeterioration of the hybridization efficiency between the probe and thedetection sequence.

Thus shown was that, when the sequence complementary to the detectionsequence overlapped the sequence of the primer annealing on thesingle-stranded loop, the primer remaining in the solution inhibited thehybridization reaction between the probe nucleic acid and the detectionsequence, prohibiting accurate measurement result. It is highly probablethat such an influence is exerted not only on the loop primer shown inthe present Example but also on the inner primer bound to thesingle-stranded loop.

As shown in FIG. 17, in the case of A where the analyte sample wasamplified with six kinds of primers containing the loop primer LFchaving a region overlapping the sequence region that was complementaryto the detection sequence placed in the F region side in the presentExample, inhibition of the hybridization between the probe nucleic acidand the target nucleic acid was observed as shown above. Alternatively,in the case of C where the analyte sample was amplified with four kindsof primers without addition of loop primer LFc or LBc, amplificationefficiency is lower, and the period needed for complete amplificationwas as long as 120 minutes.

Yet alternatively in the case of B where the analyte sample wasamplified with five kinds of primers containing a loop primer LBc placedonly in the B region where the detection sequence is not placed, therewas no hybridization inhibition, and hybridization was completed in 60minutes. The results indicated that, when a detection sequence or asequence complementary to the detection sequence and a loop primercannot be placed in the same loop, a method of placing a loop primer ina loop different from the loop containing the detection sequence or thesequence complementary to the detection sequence is effective in termsof LAMP amplification efficiency and detection efficiency of theamplification product.

Example 4 LAMP Amplification when a Primer is Placed onSingle-Nucleotide Mutation Site

In the present Example, human gene N-acetyl transferase 2 (NAT2) wasused as sample nucleic acid, and 857 G/A single-nucleotide mutationpresent in the NAT2 gene was detected (FIG. 18). 857 G/G homo-, 857 A/Ahomo- and 857 G/A-hetero genes with known sequencing were used as thehuman genomes. A detection sequence containing single-nucleotidemutation was placed in the single-stranded loop region of the NAT2 LAMPproduct, and the LAMP-amplification product under the condition shownbelow was hybridized with a probe nucleic acid immobilized on a goldelectrode. After cleaning, the detection sequence present in the LAMPamplification product was detected by a current-detecting method.

Detection Sequence

(xiii) ATAGTAAGGGATCCATCACCAGG (xiv) AAATAGTAAGGGATTCATCACCAGGT

1) Synthetic Nucleotide Primer

NAT2 F3 primer GTGGGCTTCATCCTCAC NAT2 FIP primerAGCACTTCTTCAACCTCTTCCTC(F1c)-TAAAGACAATACAGATCTGGT CG(F2) NAT2 B3 primerTGATAATTAGTGAGTTGGGTGAT NAT2 BIP primerGGGGAGAAATCTCGTGCCCAA(B1c)-GGGTTTATTTTGTTCCTTATTC (B2) NAT2 LFc primerAGTGAGAGTTTTAAACTCGACC NAT2 LBc-G primer CTGGTGATGGATCCCTTAC NAT2 LBc-Aprimer CCTGGTGATGAATCCCTTAC

The chain length of the LB-G and LB-A primers placed at thesingle-nucleotide mutation site was so adjusted that the Tm valuesthereof become almost the same.

2) LAMP Reaction Solution

The LAMP reaction solution used had a composition similar to that inExample 2. When two primers, LBc-G and LBc-A, were used together as theLBc primers, the concentration of the LBc-G and LBc-A primers arereduced to half respectively.

Amplification Reaction by LAMP Method

A reaction solution was prepared with four kinds of primers withoutthree kinds of loop primers, by using (D) human genome 857 G/G homo and(E) human genome 857 A/A homo as templates.

Separately, the following reaction solutions (F) to (I) were prepared byusing human genome 857 G/A hetero as a template: (F) solution containingseven kinds of primers containing all loop primers; (G) solutioncontaining six kinds of primers excluding LBc-A primer; (H) solutioncontaining five kinds of primers excluding LBc-G and LBc-A primers; and(I) solution containing four kinds of primers excluding three kinds ofloop primers (LFc, LBc-G, and LBc-A). Respective reaction solutions wereallowed to react at 63° C. for 30, 60, 90, or 120 minutes.

The amplification product was analyzed by agarose electrophoresis. Theamplification product in a solution containing sterile ultrapure waterreplacing the genome added during preparation of the LAMP reactionsolution was also analyzed by electrophoresis, confirming there was nocontamination.

Preparation of Probe Nucleic Acid-Immobilized Electrode

The nucleotide sequences of the probe nucleic acids used are shownbelow.

negative probe GACTATAAACATGCTTTCCGTGGCA (xiii) positive G probeCCTGGTGATGGATCCCTTACTAT (xiv) positive A probeACCTGGTGATGAATCCCTTACTATTT

The positive probes were minus chain modified 3′-terminal with SH-group.The negative probe had a sequence unrelated to the NAT2 gene sequenceand modified 3′-terminal with SH-group The probe was immobilized on agold electrode according to a method similar to that in Example 1. Thesame probe solution was also spotted on three electrodes similarly.

Electrode Allocation

Electrodes 1 to 3: negative probeElectrodes 4 to 6: positive G probeElectrodes 7 to 9: positive A probe

Preparation of Reaction Solution and Hybridization

A salt was added to the solution comprising amplification products D, E,F, G, H, and I obtained after reaction at 63° C. for 60 minutes and alsoto a mixture D+E of the amplification products D and E in the sameamount to a final concentration of 2×SSC. Separately, a solution at afinal concentration of 2×SSC was also prepared as the control. Each ofthese solutions was hybridized with the probe nucleic acid immobilizedon a gold electrode at 55° C. for 20 minutes. Then, the electrode waswashed with 0.2×SSC buffer at 45° C. for 20 minutes and then slightlywith ultrapure water. The electrode was then immersed in a phosphatebuffer solution containing 50 μm of an intercalating agent Hoechst 33258for 15 minutes, and then the oxidation current response from the Hoechst33258 molecule was measured.

Results

As shown in FIG. 19, the LAMP product obtained by using D. human genome857 G/G homo and E. human genome 857 A/A homo as the templates and noloop primer showed increase in signal from the G or A probe. Inaddition, the D+E product, artificial hetero product obtained by mixingD and E, showed increase in signal from both the G and A probes almostsimilar in intensity.

The F product obtained by using 857 G/A hetero human gene as thetemplate and seven kinds of primers including the LBC-A and LBc-Gprimers made the Tm values of the LBC-A and LBc-G primers similar toeach other, but A-typed signal was greater. The results showed that itwas difficult to balance the reaction efficiencies of the G- and A-typedprimers.

Alternatively, the LAMP reaction solution G containing six kinds ofprimers including LBc-G primer but excluding LBc-A primer showed aG-typed signal reflecting the genotype of loop primer and almost noA-typed signal. The results indicated that the G amplification productalmost did not reflect the genotype of the template, and that theproduct corresponding to the type of gene introduced into the loopprimer was amplified predominantly.

On the other hand, H and I products obtained by using no primeroverlapping the mutation site gave a signal accurately reflecting thetype of template. These results suggested that it was necessary not toplace a primer in the polymorphic (mutational) region, for obtaining anamplification product accurately reflecting the type of the template.

As shown in FIG. 20, as for the relationship between the added primerand the saturation amplification period, the product H having a loopprimer LBc annealing on the F region side without overlapping thesingle-nucleotide mutation site reached saturation of amplification in30 minutes. On the other hand, amplification for the product I with noadded loop primer needed a saturation amplification period of as long as60 minutes. The results indicated that, when a loop primer cannot beplaced in the loop containing a single-nucleotide mutation region or aregion of its complementary chain, a method of placing the loop primerin a loop different from the loop containing the single-nucleotidemutation (single-nucleotide mutation) region or the region of itscomplementary chain is effective in terms of LAMP amplificationefficiency and detection efficiency of the amplification product.

Additional advantages and modifications will readily occur to thoseskilled in the art. Therefore, the invention in its broader aspects isnot limited to the specific details and representative embodiments shownand described herein. Accordingly, various modifications may be madewithout departing from the spirit or scope of the general inventiveconcept as defined by the appended claims and their equivalents.

1. A method of designing primers for use in a method of detecting anamplification product by hybridizing it with a probe, the amplificationproduct is amplified from a target nucleic acid with the primers, on thepresupposition that: the target nucleic acid comprise F3, F2 and F1regions in this order from a 5′ terminal side, B3c, B2c and B1c regionsin this order from a 3′ terminal side, as well as an FP region in theregion from the F2 to F1 regions and/or a BPc region in the region fromthe B2c to B1c regions; the probe represents a probe nucleic acidcomprising a nucleotide sequence complementary to a sequence selectedfrom the group consisting of the sequence in the FP and BPc region andthe sequences complementary thereto; and the primers contain an FIPprimer comprising the sequence identical to that of the F2 region at the3′ terminal side and a sequence complementary to that of the F1 regionat the 5′ terminal side; an F3 primer comprising the sequence identicalto that of the F3 region; a BIP primer comprising a sequencecomplementary to that of the B2c region at the 3′ terminal side and thesequence identical to that of the B1c region at the 5′ terminal side;and a B3 primer comprising a sequence complementary to that of the B3cregion: the method comprising setting the respective regions in such amanner that the FP and F2 regions and/or the BPc and B2c regions have anunoverlapping region of at least 10 bases or more and overlappingregions of 10 bases or less, and designing the primers according to theregions.
 2. The method according to claim 1, on the presupposition that:the target nucleic acid further comprise an LF region in the region fromthe F2 to F1 regions and/or an LBc region in the region from the B2c toB1c regions; and the primers contain the FIP primer, the F3 primer, theBIP primer, and the B3 primer, as well as a loop primer LFc comprising asequence complementary to that of the LF region and/or a loop primer LBccomprising the sequence identical to that of the LBc region: the methodfurther comprising setting the respective regions in such a manner thatthe FP and LF regions and/or the BPc and LBc regions have anunoverlapping region of at least 10 bases or more and overlappingregions of 10 bases or less.
 3. A method of detecting a target nucleicacid, comprising a step of obtaining an amplification product byamplifying a target nucleic acid with primers and a step of detectingthe amplification product by hybridizing it with a probe, on thepresupposition that: the target nucleic acid comprise F3, F2 and F1regions in this order from a 5′ terminal side, B3c, B2c and B1c regionsin this order from a 3′ terminal side, as well as an FP region in theregion from the F2 to F1 regions and/or a BPc region in the region fromthe B2c to B1c regions; the probe represents a probe nucleic acidcomprising a nucleotide sequence complementary to a sequence selectedfrom the group consisting of the sequence in the FP and BPc region andthe sequences complementary thereto; and the primers contain an FIPprimer comprising the sequence identical to that of the F2 region at the3′ terminal side and a sequence complementary to that of the F1 regionat the 5′ terminal side; an F3 primer comprised the sequence identicalto that of the F3 region; a BIP primer comprising a sequencecomplementary to that of the B2c region at the 3′ terminal side and thesequence identical to that of the B1c region at the 5′ terminal side;and a B3 primer comprising a sequence complementary to that of the B3cregion: the FP and F2 regions and/or the BPc and B2c region have anunoverlapping region of at least 10 bases or more and overlappingregions of 10 bases or less.
 4. The method according to claim 3, whereinthe probe nucleic acid is immobilized on a solid-phase support.
 5. Themethod according to claim 4, wherein the solid-phase support is a DNAchip.
 6. The method according to claim 3, on the presupposition that:the target nucleic acid further comprise an LF region in the region fromthe F2 to F1 regions and/or an LBc region in the region from the B2c toB1c regions; and the primers used in the amplification step are the FIPprimer, the F3 primer, the BIP primer, and the B3 primer, as well as aloop primer LFc comprising a sequence complementary to that of the LFregion and/or a loop primer LBc comprising the sequence identical tothat of the LBc region: the FP and LF regions and/or the BPc and LBcregions have an unoverlapping region of at least 10 bases or more andoverlapping regions of 10 bases or less.
 7. The method according toclaim 6, wherein the probe nucleic acid is immobilized on a solid-phasesupport.
 8. The method according to claim 7, wherein the solid-phasesupport is a DNA chip.
 9. A method of designing primers for use in amethod of detecting a single-nucleotide mutation by amplifying a targetnucleic acid having the single-nucleotide mutation with primers andhybridizing the amplification product with a probe, on thepresupposition that: the target nucleic acid comprise F3, F2 and F1regions in this order from a 5′ terminal side, B3c, B2c and B1c regionsin this order from a 3′ terminal side, as well as an FP region havingthe single-nucleotide mutation in the region from the F2 to F1 regionsand/or a BPc region having the single-nucleotide mutation in the regionfrom the B2c to B1c regions; the probe represents a probe nucleic acidcomprising a nucleotide sequence complementary to a sequence selectedfrom the group consisting of the sequence in the FP and BPc region, andthe sequences complementary thereto; and, the primers contain an FIPprimer comprising the sequence identical to that of the F2 region at the3′ terminal side and a sequence complementary to that of the F1 regionat the 5′ terminal side; an F3 primer comprising the sequence identicalto that of the F3 region; a BIP primer comprising a sequencecomplementary to that of the B2c region at the 3′ terminal side and thesequence identical to that of the B1c region at the 5′ terminal side;and a B3 primer comprising a sequence complementary to that of the B3cregion: the method comprising setting the F2 region not to overlap thesingle-nucleotide mutation in the FP region and/or the B2c region not tooverlap the single-nucleotide mutation in the BPc region, and designingthe primers according to the regions.
 10. The method according to claim9, on the presupposition that: the target nucleic acid further comprisean LF region in the region from the F2 to F1 regions and/or an LBcregion in the region from the B2c to B1c regions; and the primerscontain the FIP primer, the F3 primer, the BIP primer, and the B3primer, as well as a loop primer LFc comprising a sequence complementaryto that of the LF region and/or a loop primer LBc comprising thesequence identical to that of the LBc region: the method furthercomprising setting the LF region not to overlap the single-nucleotidemutation in the FP region and/or the LBc region not to overlap thesingle-nucleotide mutation in the BPc region, and designing the primersaccording to the regions.
 11. A method of detecting a single-nucleotidemutation, comprising: a step of obtaining an amplification product byamplifying a target nucleic acid having the single-nucleotide mutationwith primers; and a step of detecting the amplification product byhybridizing it with a probe, on the presupposition that: the targetnucleic acid comprise F3, F2 and F1 regions in this order from a 5′terminal side, B3c, B2c and B1c regions in this order from a 3′ terminalside, as well as an FP region having the single-nucleotide mutation inthe region from the F2 to F1 regions and/or a BPc region having thesingle-nucleotide mutation in the region from the B2c to B1c regions;the probe represents a probe nucleic acid comprising nucleotide sequencecomplementary to a sequence selected from the group consisting of thesequence in the FP and BPc region, and the sequences complementarythereto; and the primers contain an FIP primer comprising the sequenceidentical to that of the F2 region at the 3′ terminal side and asequence complementary to that of the F1 region at the 5′ terminal side;an F3 primer comprising the sequence identical to the F3 region; a BIPprimer comprising a sequence complementary to that of the B2c region atthe 3′ terminal side and the sequence identical to that of the B1cregion at the 5′ terminal side; and a B3 primer comprising a sequencecomplementary to that of the B3c region: the F2 region does not overlapthe single-nucleotide mutation in the FP region and/or the B2c regiondoes not overlap the single-nucleotide mutation in the BPc region. 12.The method according to claim 11, wherein the probe nucleic acid isimmobilized on a solid-phase support.
 13. The method according to claim12, wherein the solid-phase support is a DNA chip.
 14. The methodaccording to claim 11, on the presupposition that: the target nucleicacid further comprise an LF region in the region from the F2 to F1regions and/or an LBc region in the region from the B2c to B1c regions;and the primers used in the amplification step are the FIP primer, theF3 primer, the BIP primer and the B3 primer, as well as a loop primerLFc comprising a sequence complementary to that of the LF region and/ora loop primer LBc comprising the sequence identical to that in the LBcregion: the LF region does not overlap the single-nucleotide mutation inthe FP region and/or the LBc region does not overlap thesingle-nucleotide mutation in the BPc region.
 15. The method accordingto claim 14, wherein the probe nucleic acid is immobilized on asolid-phase support.
 16. The method according to claim 15, wherein thesolid-phase support is a DNA chip.
 17. An assay kit for use in a methodof detecting an amplification product by hybridizing it with a probe,which the amplification product is amplified from a target nucleic acidwith primers, on the presupposition that: the target nucleic acidcomprise F3, F2 and F1 regions in this order from a 5′ terminal side andB3c, B2c and B1c regions in this order from a 3′ terminal side, as wellas an FP region in the region from the F2 to F1 regions and/or a BPcregion the region from the B2c to B1c regions; the probe represents aprobe nucleic acid comprising a nucleotide sequence complementary to asequence selected from the group consisting of the sequence in the FPand BPc region and the sequences complementary thereto; and the primerscontain an FIP primer comprising the sequence identical to that of theF2 region at the 3′ terminal side and a sequence complementary to thatof the F1 region at the 5′ terminal side; an F3 primer comprising thesequence identical to that of the F3 region; a BIP primer comprising asequence complementary to that of the B2c region at the 3′ terminal sideand the sequence identical to that of the B1c region at the 5′ terminalside; and B3 primer comprising a sequence complementary to that of theB3c region: the assay kit comprising the respective primers designedaccording to the respective regions set in such a manner that the FP andF2 regions and/or the BPc and B2c regions have an unoverlapping regionof at least 10 bases or more and overlapping regions of 10 bases orless, as well as a buffer, a strand-displacing DNA polymerase, and adNTP.
 18. The assay kit according to claim 17, on the presuppositionthat: the target nucleic acid further comprising an LF region in theregion from the F2 to F1 regions and/or an LBc region in the region fromthe B2c to B1c regions; and the primers contain the FIP primer, the F3primer, the BIP primer, and the B3 primer, as well as a loop primer LFccomprising a sequence complementary to that of the LF region and/or aloop primer LBc comprising the sequence identical to that of the LBcregion: the assay kit further comprising the respective primers designedaccording to the respective regions set in such a manner that the FP andLF regions and/or the BPc and LBc regions have an unoverlapping regionof at least 10 bases or more and overlapping regions of 10 bases orless.
 19. An assay kit for use in a method of detecting asingle-nucleotide mutation by amplifying a target nucleic acid havingthe single-nucleotide mutation with primers and hybridizing theamplification product with a probe, on the presupposition that: thetarget nucleic acid comprise F3, F2 and F1 regions in this order from a5′ terminal side and B3c, B2c and B1c regions in this order from a 3′terminal side, as well as an FP region containing the single-nucleotidemutation in the region from the F2 to F1 regions and/or a BPc regioncontaining the single-nucleotide mutation in the region from the B2c toB1c regions; the probe represents a probe nucleic acid comprisingnucleotide sequence complementary to a sequence selected from the groupconsisting of the sequence in the FP and BPc region, and the sequencescomplementary thereto; and the primers contain an FIP primer comprisingthe sequence identical to that of the F2 region at the 3′ terminal sideand a sequence complementary to that of the F1 region at the 5′ terminalside; an F3 primer comprising the sequence identical to that of the F3region; a BIP primer comprising a sequence complementary to that of theB2c region at the 3′ terminal side and the sequence identical to that ofthe B1c region at the 5′ terminal side; and B3 primer comprising asequence complementary to that of the B3c region: the assay kitcomprising the respective primers designed according to the respectiveregions set in such a manner that the F2 region does not overlap thesingle-nucleotide mutation in the FP region and/or the B2c region doesnot overlap the single-nucleotide mutation in the BPc region, as well asa buffer, a strand-displacing DNA polymerase, and a dNTP.
 20. The assaykit according to claim 19, on the presupposition that: the targetnucleic acid further comprising an LF region in the region from the F2to F1 regions and/or an LBc region in the region from the B2c to B1cregions; and the primers contain the FIP primer, the F3 primer, the BIPprimer and the B3 primer, as well as a loop primer LFc comprising asequence complementary to that of the LF region and/or a loop primer LBccomprising the sequence identical to that of the LBc region: the assaykit further comprising the respective primers designed according to therespective regions set in such a manner that the LF region doesn'toverlap the single-nucleotide mutation in the FP region and/or the LBcregion doesn't overlap the single-nucleotide mutation in the BPc region.